PROJECT SUMMARY Human noroviruses (HNoVs) are medically important non-enveloped positive-sense RNA viruses that cause gastroenteritis. This revised proposal leverages the recent discovery of CD300lf as a receptor for murine NoV (MNoV, Science 2016 353:933) to define mechanisms of NoV pathogenesis, persistence and intestinal immunity. HNoVs do not robustly replicate in small animals and so murine MNoV has emerged as a model system to discover mechanisms of NoV biology and principles of intestinal immunity that would be difficult to identify in human studies or using animals that are not easily manipulated genetically. Studies utilizing MNoV have delineated important aspects of the interplay between virus and host in the intestine. While our proposal was initially restricted to MNoV, seminal work from the Estes lab demonstrates for the first time robust replication of HNoVs in human enteric enteroids and a role for bile acids in HNoV growth (see letter, Science 2016 353:1387) allowing us to collaborate with the Estes laboratory to perform HNoV studies. New data provided in Aim 1 show that specific bile acids function as the soluble 'co-factor' that we reported as essential for the function of CD300lf as an MNoV receptor1. These data suggest a remarkable conservation of dependence on a host factor present in the intestine for both HNoV and MNoV. Based on these advances we propose new studies herein and believe that these studies will enhance the value of our proposed work. NoV species and cell tropism is determined at the level of cell binding and entry. Thus, host factors including receptor(s) required for infection and pathogenesis are incompletely understood for this viral genus, creating key knowledge gaps for understanding NoV spread, pathogenesis, vaccination, species tropism and persistence. To identify host molecules required for MNoV infection we performed a CRISPR-Cas9-based screen, and discovered that CD300lf is a receptor for MNoV in primary cells and BV2 microglial cells and that it is required for infection in vivo. CD300lf confers the ability of MNoV to bind to cells at 4oC, and is sufficient to render human HeLa cells fully permissive for MNoV replication. The crystal structure of the CD300lf ectodomain revealed an Ig-domain with a ligand binding pocket; amino acids comprising this binding pocket are critical for receptor function for MNoV infection. The soluble ectodomain (sCD300lf) neutralized viral infectivity in vitro and in vivo and we now show that it directly binds to the virion. A soluble cofactor was required for CD300lf receptor function, and new experiments show that specific bile acids function as soluble cofactors. Given these data we propose to test the hypothesis that CD300lf confers tissue and cell tropism responsible for the capacity of MNoV to establish persistent intestinal infection and to elicit immunity through the following Aims: Aim 1: Determine the molecular basis of the role of CD300lf in NoV infection. Aim 2: Determine the role of CD300lf in MNoV tropism, persistence and immunity.